DESCRIPTION Small cell lung cancer (SCLC) remains a significant clinical problem that affects both men and women and causes an estimated 30,000 patient deaths every year in the US. The high mortality rate of this cancer is mainly due to the difficulty of early detection and the inadequacy of current genotoxic chemotherapies that have remained largely unchanged for the past 30 years. It is essential that new preventive and therapeutic approaches are developed to improve patient outcomes. Functional characterization of recurrent mutations in the SCLC genome will facilitate discovery of biomarkers for prevention and targeted therapy, but this remains extremely challenging due to the paucity of robust experimental models. Genetically engineered mouse models (GEMM) of human SCLC have been important tools for determining functions of oncogenes and tumor suppressor genes and evaluating therapeutic treatments. However, utilization of the GEMMs requires laborious and expensive genetic crosses to test the function of even a single gene. To overcome these challenges, we have developed an in vitro tumor progression model to define oncogenic function of CRISPR-mediated mutations based on their ability to transform precancerous cells (preSC) which were isolated from a genetically engineered mouse model of SCLC that carries conditional alleles of Rb and p53, the most frequently mutated genes in human SCLC. Comparative profiling of mutant preSC transformed by a candidate mutation with control preSC allows us to distinguish mutation-specific genes and pathways from a multitude of secondary or adaptive changes. We have also developed a somatic engineering-based model in which an adenoviral CRISPR-Cre hybrid vector, delivered directly in the lung airways, allows us to characterize CRISPR-mediated mutation during in vivo tumor development in conjunction with Cre-mediated inactivation of Rb and p53. This new in vivo approach enables rapid functional interrogation of candidate mutations in vivo without need for expensive, time-consuming genetic crosses. Using these complementary models, we will test the hypothesis that recurrent mutations in SCLC are sufficient to cause the tumorigenic progression of Rb/p53-mutant precursor cells. In aim 1, we will discover mutation-driven oncogenic pathways using comparative analysis of engineered precancerous cells. In aim 2, we will use the CRISPR-Cre hybrid vector to characterize novel mutations in tumor development in vivo. Successful completion of this proposal will define a dozen of the most recurrent mutations in SCLC and provide preclinical models of SCLC carrying specific sets of mutations. These outcomes will be groundbreaking for future studies aimed at mechanistic elucidation of the tumor development as well as development of preventive and therapeutic strategies for this cancer. Moreover, the CRISPR-Cre hybrid tools are easily extendable to test more mutations and are readily applicable to other cancer models that are receptive to adenoviral gene delivery, including those of non-small cell lung cancers.